7. Project Summary/Abstract Altered metabolism is a nearly universal characteristic of cancer cells, which require metabolic adaptations to support proliferation and survival. However, mechanisms that dictate the diverse metabolic liabilities and phenotypes of cancer remain poorly understood. Thus, the resurgence of interest to target cancer metabolism for patient benefit will require an improved understanding of metabolic regulation and requirements in malignant cell types. Cells in culture are commonly used to study cancer metabolism and to develop drugs that exploit metabolic vulnerabilities. However, while it is increasingly appreciated that environmental factors impact cell metabolism, our current understanding of metabolic rewiring in cancer is largely based on findings from cells cultured in media that poorly reflect the metabolic composition of human plasma. Therefore, our overarching hypothesis is that many important aspects of cancer cell metabolism have been overlooked or misconstrued as a consequence of utilizing model systems that inadequately resemble physiologic conditions. To begin to test this, we developed a new culture medium (human plasma-like medium; HPLM) that contains polar metabolites and salts at concentrations comparable to those of adult human plasma. We then showed that, relative to traditional media, HPLM has widespread and largely unexplained effects on cell metabolism. Our preliminary data reveal that, when cultured in traditional media, certain cell lines secrete alanine, but that in HPLM, those cells instead consume alanine at rates exceeding those for most other amino acids. Therefore, we will test the hypothesis that alanine has a key role for cells cultured in physiologic conditions (Aim 1). Using a chemostat technology that we developed, we will also determine how the removal of alanine from HPLM affects the growth of over 40 barcoded blood cancer cell lines in a pooled fashion. In addition, through the use of CRISPR-based loss-of-function screens, we identified NME6, a gene that encodes a poorly studied mitochondrial protein, as conditional lethal with HPLM. Thus, we will also test the hypothesis that NME6 serves a critical and unforeseen role in mitochondrial homeostasis for cells cultured in physiologic conditions (Aim 2). Lastly, we also found that HPLM dramatically influences cell sensitivity to the cancer drug 5-fluorouracil without affecting the potency of doxorubicin, another common chemotherapeutic. Therefore, using a library of > 1,900 diverse oncology-related small molecules, we will perform a high-throughput screen to test the hypothesis that, relative to traditional media, HPLM alters the potency of additional compounds. We will then pursue validation and mechanistic insights for differential drug phenotypes in cell line and primary cell models (Aim 3). Our proposed work uses distinct approaches to better understand how environmental factors that more closely reflect physiologic conditions impact the metabolism of blood cancer cells, and has the potential to not only identify unforeseen biological insights, but to also uncover new therapeutic targets and approaches that may have greater in vivo relevance for cancer therapy.